Collagenase type 2 digestion

Manufactured by Merck Group

Sourced in United States

Collagenase type II is an enzyme used for the digestion of collagen, a structural protein found in the extracellular matrix. It is commonly used in various cell isolation and tissue dissociation applications.

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Lab products found in correlation

Nylon cell strainer, by Merck Group (3 mentions) Ficoll hypaque, by Pfizer (3 mentions) 70 μm nylon cell strainer, by BD (2 mentions) 70 m nylon cell strainer, by BD (1 mentions) Magnetic bead, by Miltenyi Biotec (1 mentions)

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Collagenase (2150 citations) Collagenase type II (832 citations) Collagenase II (460 citations) Type II (13 citations) Collagenase digestion (12 citations) Type collagenase (3 citations)

4 protocols using collagenase type 2 digestion

Isolation of Immune Cells from Clinical Samples

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Samples of blood, endocervical curettage and endometrial biopsies collected at UCSF were transported on ice to UC Davis to arrive within 3–4 hours of collection. Upon arrival, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Pfizer, New York, NY) density gradient centrifugation, and washed in PBS. Mononuclear cells from endocervical curettage were isolated by repeated pipetting, followed by passage through a 70μm nylon cell strainer (Becton Dickinson, Bedford, MA) and washing in complete medium [RPMI-1640 supplemented with 15% FBS, penicillin (100U/ml), streptomycin (100μg/ml), and glutamine (2mM)]. Endometrial biopsies were subjected to two to three rounds of collagenase type II digestion (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO), followed by mechanical disruption using an 18-gauge blunt-end needle and passage through a 70-μm nylon cell strainer. The pooled cells were washed in complete medium.
Red blood cell lysis was performed as needed on PBMC, endometrium and endocervical cells using ammonium chloride-potassium carbonate-EDTA (ACK).

Smith-McCune K., Chen J.C., Greenblatt R.M., Shanmugasundaram U., Shacklett B.L., Hilton J.F., Johnson B., Irwin J.C, & Giudice L.C. (2015). Unexpected Inflammatory Effects of Intravaginal Gels (Universal Placebo Gel and Nonoxynol-9) on the Upper Female Reproductive Tract: A Randomized Crossover Study. PLoS ONE, 10(7), e0129769.

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Isolation of Immune Cells from Clinical Samples

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Samples of blood, endocervical brushings, and endometrial biopsies collected at UCSF were transported on ice to UC Davis to arrive within 3–4 hr of collection (Figs 2–4). Upon arrival, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque (Pfizer, New York, NY, USA) density gradient centrifugation, and washed in PBS. Mononuclear cells from endocervical brushings were isolated by rubbing the brushes together, pipetting several times, passing through a 70-µm nylon cell strainer (Becton Dickinson, Bedford, MA, USA) and washing in complete medium [RPMI-1640 supplemented with 15% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), and glutamine (2 mmol/L)]. Endometrial biopsies were subjected to 2–3 rounds of collagenase type II digestion (0.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA), followed by mechanical disruption using an 18-gauge blunt-end needle and passage through a 70-µm nylon cell strainer. The pooled cells were washed in complete medium. Red blood cell lysis was performed as needed on PBMC, endometrium and endocervical cells using ammonium chloride–potassium carbonate–EDTA (ACK).

Shanmugasundaram U., Hilton J.F., Critchfield J.W., Greenblatt R.M., Giudice L.C., Averbach S., Seidman D., Shacklett B.L, & Smith‐McCune K. (2016). Effects of the levonorgestrel‐releasing intrauterine device on the immune microenvironment of the human cervix and endometrium. American Journal of Reproductive Immunology, 76(2), 137-148.

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Enrichment of Mouse and Rat SSCs

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Testis cells were dissociated by collagenase type II digestion (1 mg/ml; Sigma), as described previously [21 (link)]. For enrichment of mouse SSCs,
the dissociated testis cells were incubated with an anti-CDH1 antibody (ECCD2; gift from Dr M Takeichi from RIKEN CDB, Kobe, Japan), and cells were isolated using magnetic beads,
as described previously (Miltenyi Biotec, Gladbach, Germany) [14 (link)]. For enrichment of rat SSCs, we used an anti-CD9 antibody (RPM.7; BD
Biosciences, Franklin Lakes, CA, USA).

KANATSU-SHINOHARA M., CHEN G., MORIMOTO H, & SHINOHARA T. (2020). CD2 is a surface marker for mouse and rat spermatogonial stem cells. The Journal of Reproduction and Development, 66(4), 341-349.

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Isolation of Peripheral Blood, Endocervical, and Endometrial Cells

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Samples of blood, endocervical brushings, and endometrial biopsies collected at UCSF were transported on ice to UC Davis to arrive within 3–4 hr of collection (Figs 2, 3, 4). Upon arrival, peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque (Pfizer, New York, NY, USA) density gradient centrifugation, and washed in PBS. Mononuclear cells from endocervical brushings were isolated by rubbing the brushes together, pipetting several times, passing through a 70‐μm nylon cell strainer (Becton Dickinson, Bedford, MA, USA) and washing in complete medium [RPMI‐1640 supplemented with 15% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), and glutamine (2 mmol/L)]. Endometrial biopsies were subjected to 2–3 rounds of collagenase type II digestion (0.5 mg/mL; Sigma‐Aldrich, St. Louis, MO, USA), followed by mechanical disruption using an 18‐gauge blunt‐end needle and passage through a 70‐μm nylon cell strainer. The pooled cells were washed in complete medium. Red blood cell lysis was performed as needed on PBMC, endometrium and endocervical cells using ammonium chloride–potassium carbonate–EDTA (ACK).

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